Within the bodies of mated female mammals and insects, seminal proteins cause biochemical and physiological effects that are important for fertility. Despite their importance, the functions of these proteins are not well understood. We use Drosophila's Acp seminal proteins as a model to investigate the mechanisms by which seminal proteins affect females. Acps fall into biochemical classes that are conserved across animals and regulate several analogous reproductive phenomena across animals. Thus, our use of Drosophila genetics to dissect seminal protein function informs the understanding of human fertility and the control of insect vectors of disease. Through genetic tests of over 40 Acps and molecular tests of their function, we have identified individual Acps that regulate specific steps in reproductive processes. We have shown that their action requires molecular or physiological crosstalk between male-derived Acps and molecules or conditions in females. To define precisely how specific Acps work together with the female to mediate their effects, we propose to focus on two steps that occur rapidly after mating in Drosophila. Specifically, we will dissect the role of the Acp ovulin in triggering ovulation by determining its site of action, the role of its processing, the identity of its receptor, and whether ovulin mediates muscle contractions and acts with the biogenic amine octopamine. We will dissect the way in which the seminal protein Acp36DE promotes the entry of sperm into storage by determining its active regions, its receptor, and whether it triggers vesicle release and acts through particular female reproductive tract proteins to cause conformational changes in the female's reproductive tract. Finally, we will define how a proteolytic pathway composed of male- and female-derived components processes ovulin within mated females. Relevance: The seminal proteins of the fruit fly Drosophila are similar in type to those found in people and other animals, and they cause reproductive effects that are analogous across organisms. This allows us to take advantage of the rapid and unusually powerful genetics of Drosophila to quickly and precisely determine how seminal proteins work to facilitate reproduction. Because of the parallels in seminal protein actions across animals, our results will provide information useful to interpret and address the causes of certain human infertilities, and will help to design methods to control the spread of insects that transmit diseases to people.